Human embryonic stem cells provide a potentially unlimited source of oral mucosal tissues that can revolutionize the treatment of oral diseases. These stem cells are essential to the success of novel regenerative therapies, due to their plasticity and to their tremendous regenerative potential that can overcome the shortcomings and currently limited supply of post-natal, oral epithelial stem cells. However, many obstacles still limit their therapeutic application, as it is not known if human embryonic stem cells can 1- form 3D epithelial tissues with hierarchical structure, 2 - give rise to cells similar to post-natal, epithelial stem cells or 3 - maintain their differentiated state in 3D tissues. To help overcome these barriers, the immediate goal of this application is to use our novel approaches in 3D tissue biology to provide key insights into the biological properties of human embryonic stem cells to generate 3D oral epithelial tissues that will demonstrate the feasibility of using these new technologies for future oral therapies. We hypothesize that the tissue microenvironment, instructed by basement membrane proteins and fibroblast-derived soluble factors, regulates the self-renewal and differentiation of keratinocytes derived from human embryonic stem cells to generate specialized tissues that mimic oral epithelium. To test this hypothesis, our AIMS are to: 1- Determine if keratinocytes derived from human embryonic stem cells give rise to progenitor cells with properties of post-natal stem cells that can undergo long-term, self-renewal and can differentiate to form 3D epithelia and 2- Begin to explore the cellular- and tissue-based cues, mediated by the stem cell "niche" at the basement membrane interface and by fibroblast-derived soluble factors, that direct these events. We expect to set the stage for future studies that will dissect signaling pathways that direct these cues and optimize the growth, differentiation and survival of 3D epithelia. This research is made possible by two major advances in our lab: 1 - Development of engineered, 3D tissues that mimic the oral mucosa to a significant degree and have established a link between the tissue microenvironment, self- renewal of post-natal stem cells and predictable tissue regeneration after in vivo transplantation. 2 - Propagation of H9, human embryonic stem cells (NIH-WA09) committed to an epithelial lineage and generation of immature but multilayer 3D epithelial tissues from their progeny. Our long-term goal is to harness the developmental potential and growth capacity of pluripotent, human embryonic stem cells by gaining convincing evidence that oral epithelial tissues derived from them can serve as long-term, functional replacement tissues to treat oral diseases for which there are no current practical therapies. By doing so, we will advance our understanding of stem cell biology in a fundamental manner to enable future efforts to tailor and engineer lineage-specific tissues of therapeutic importance in humans.